The characterization of a concentration-sensitive α-adrenergic-like octopamine receptor found on insect immune cells and its possible role in mediating stress hormone effects on immune function

https://doi.org/10.1016/j.bbi.2012.04.007Get rights and content

Abstract

Octopamine (OA), the insect equivalent of norepinephrine, links the nervous system and immune system in insects. This study examines the underlying molecular mechanisms (i.e. second messenger systems) mediating OA effects on insect immune cells. At low concentrations (<1 μM), OA stimulated hemocyte spreading and phagocytosis in the larval Lepidopteran (caterpillar) Chilo suppressalis, whereas at high concentrations (>10 μM), OA inhibited hemocyte spreading and phagocytosis. Similarly, OA concentration had differential effects on two intracellular signaling pathways, Ca2+ and cAMP. Low concentrations of OA increased intracellular Ca2+, but only high concentrations of OA (>1 μM) led to an increase in both Ca2+ and cAMP. We identified an α-adrenergic-like octopamine receptor in this species (CsOA1) and confirmed that it is expressed in hemocytes. After heterologous expression in HEK-293 cells, the CsOA1 receptor produced the same OA concentration-dependent responses on intracellular Ca2+ and cAMP as had been observed in hemocytes. These findings support earlier work showing that OA has both stimulatory and suppressive effects on immune responses, depending on the OA concentration. Our evidence suggests that these biphasic effects are mediated by an octopamine receptor signaling through intracellular Ca2+ and cAMP second messenger pathways. Stress hormones/neuromodulators have complex effects on immune function in animals across phyla. This complexity may be mediated, in part, by conserved connections between adrenergic-like G-coupled protein receptors and second messenger systems.

Highlight

► First molecular demonstration of octopamine receptors in an insect immune cell suggests that the neural-immune connections may be conserved from insects to mammals.

Introduction

The nervous system regulates the immune system in vertebrates (Sternberg, 2006, Tracey, 2009). However, the effects of neurally-derived molecules (e.g. neurohormones) on immune cells, and the importance of these effects on immune system function, are still controversial (Nance and Sanders, 2007). For example, some studies show that norepinephrine, released by the sympathetic nervous system during the stress response, increases immune and pro-inflammatory responses (Garcia et al., 2003, Johnson et al., 2005, Lang et al., 2003, Straub et al., 2000) but other studies find that it causes immunosuppressive effects (Alves et al., 2006, Woiciechowsky et al., 1998). In part these controversies exist because the underlying molecular mechanisms are incompletely understood (Franco et al., 2007, Sternberg, 2006, Tracey, 2009).

Compounds released during the stress response also have complex effects on immune function in invertebrates (Adamo, 2008). Octopamine, the insect equivalent of norepinephrine (Roeder, 1999), enhances phagocytosis by insect immune cells (i.e. hemocytes) (Baines et al., 1992, Baines and Downer, 1994, Kim et al., 2009), but it can also reduce immune function and disease resistance (Adamo and Parsons, 2006, Adamo, 2010). Unfortunately the molecular mechanisms underlying OA-mediated immunoregulation of insect hemocytes are poorly understood, making this apparent contradiction difficult to resolve. OA is thought to induce these effects via octopamine receptors (OARs) located on insect immune cells (Adamo, 2008), although the molecular evidence for their existence on these cells is lacking. In fact, there are no published molecular studies to-date to verify earlier pharmacological work suggesting that receptors for stress hormones are widespread in invertebrate immune cells (Adamo, 2008).

Understanding how octopamine induces complex effects in insect immune cells may shed light on how norepinephrine produces similarly complex effects in vertebrate immune cells. The octopaminergic and noradrenergic systems are thought to have evolved from the same ancestral system because of the molecular similarities between norepinephrine and octopamine, as well as between their receptors and transporters (Evans and Maqueira, 2005). For example, the octopamine receptor is a typical rhodopsin-like G protein-coupled receptor that has a sequence similar to that of vertebrate adrenergic receptors (Evans and Maqueira, 2005). In cells outside of the immune system, the binding of OA to OARs has been shown to activate coupled G-protein effector pathways, thereby inducing the generation of intracellular second messengers such as cAMP and/or Ca2+ (Roeder, 2005). Vertebrate adrenergic receptors are also known to activate multiple second messenger systems in some vertebrate cell types (e.g. Cotecchia et al., 1990), although, as in insects, this issue has not been well studied in immune cells. If insect hemocytes have adrenergic-like receptors, it may be possible to test whether these receptors are capable of mediating complex responses to stress hormones/neuromodulators. This information is likely to be relevant to vertebrate immune cells as well.

In the present study, we provide molecular evidence that hemocytes of the striped stem borer (SSB), Chilo suppressalis (Walker) (Lepdiopter: Crambidae) carry an OA receptor (CsOA1) on their hemocytes. We then demonstrate that these immune cells show complex responses to stress hormone OA. We test whether some of this complexity is mediated via different second messenger systems activated by the OAR.

Section snippets

Insects

The larvae of the striped stem borer (SSB), C. suppressalis were collected from fields in Fuyang (30°3′58.93″N, 119°55′49.95″E), Zhejiang Province, China, in 2010. The collected SSB larvae were reared on an artificial diet (Liu et al., 2008) at 28 °C under a 16:8 L:D photoperiod for several generations prior to experiments.

Hemocyte-spreading assay

The in vitro bioassay was modified from an established method (Clark et al., 1997). Fifth-instar larvae of SSB were surface-sterilized with 70% ethanol. The proleg was cut

OA showed biphasic effects on cellular immune responses

10 nM and 100 nM OA enhanced hemocyte spreading significantly (one-way ANOVA, F(6, 20) = 28.8; 10−8 M, t = 5.0, p = 0.004; 10−7 M, t = 5.1, p = 0.003), but above 10 μM, OA inhibited the spreading behavior of hemocytes (Fig. 1, Fig. 2, Fig. 3, Fig. 4, 10−4 M, t = −4.2, p = 0.02). Similar results were also found in the hemocyte-phagocytosis assay (Fig. 1B), with concentrations of OA up to 10−7 M enhancing phagocytosis (one-way ANOVA, F(6, 20) = 74.5, t = 6.5, p = 0.0003), but higher concentrations (>10−5 M) inhibiting it (10−5

Discussion

The effect of OA on immune function was concentration-dependent. Low concentrations of OA promoted hemocyte spreading and phagocytosis, probably mediated by increasing intracellular calcium levels. However, high concentrations of OA inhibited hemocyte spreading and phagocytosis via the cAMP pathway. Thus, the immunoregulatory effects of OA on immune function might be different, depending on the amount of OA released.

Earlier studies have also found that OA has biphasic effects on immune

Acknowledgments

Financial support for this study was provided by the National High-tech R&D Program of China (2011AA10A204), National Natural Science Foundation of China (31000849), Qianjiang Talent Program of Zhejiang Province (2010R10086) and NSERC (Natural Sciences and Engineering Research Council of Canada).

References (45)

  • W.L. Diehl-Jones et al.

    Monoaminergic regulation of hemocyte activity

    J. Insect Physiol.

    (1996)
  • G.B. Dunphy et al.

    Octopamine, a modulator of the haemocytic nodulation response of non-immune Galleria mellonella larvae

    J. Insect Physiol.

    (1994)
  • R. Franco et al.

    The emergence of neurotransmitters as immune modulators

    Trends Immunol.

    (2007)
  • G. Grynkiewicz et al.

    A new generation of Ca2+ indicators with greatly improved fluorescence properties

    J. Biol. Chem.

    (1985)
  • K. Lang et al.

    Neurotransmitters regulate the migration and cytotoxicity in natural killer cells

    Immunol. Lett.

    (2003)
  • D. Marin et al.

    Cyclic AMP affects the haemocyte responses of larval Galleria mellonella to selected antigens

    J. Insect Physiol.

    (2005)
  • D.M. Nance et al.

    Autonomic innervation and regulation of the immune system (1987–2007)

    Brain Behav. Immun.

    (2007)
  • T. Roeder

    Octopamine in invertebrates

    Prog. Neurobiol.

    (1999)
  • S. Tojo et al.

    Involvement of both granular cells and plasmatocytes in phagocytic reactions in the greater wax moth, Galleria mellonella

    J. Insect Physiol.

    (2000)
  • H. Verlinden et al.

    The role of octopamine in locusts and other arthropods

    J. Insect Physiol.

    (2010)
  • H. Verlinden et al.

    The cloning, phylogenetic relationship and distribution pattern of two new putative GPCR-type octopamine receptors in the desert locust (Schistocerca gregaria)

    J. Insect Physiol.

    (2010)
  • S.A. Adamo

    Parasitic suppression of feeding in the tobacco hornworm, Manduca sexta: parallels with feeding depression after an immune challenge

    Arch. Insect Biochem. Physiol.

    (2005)
  • Cited by (54)

    • Molecular characterization and functional roles for Vibrio alginolyticus resistance of an octopamine/tyramine receptor of the white shrimp, Litopenaeus vannamei

      2022, Fish and Shellfish Immunology
      Citation Excerpt :

      However, OA released from RIC neurons activates OA receptor-1 (OCTR-1) that functions in the sensory ASH neurons to inhibit innate immune responses in nonneural tissues in C. elegans [61]. CsOA1 in C. suppressalis acts through both Ca2+ and cAMP signaling pathways to enhance hemocytes spreading and phagocytosis at low OA concentration (10−7 and 10−8 M) but inhibit hemocytes spreading and phagocytosis at high OA concentration (10−3 and 10−4 M) [22]. It appears that OA receptor can modulate opposite immune responses effect in different species, and display multiple function depending on dosage.

    • How insects protect themselves against combined starvation and pathogen challenges, and the implications for reductionism

      2021, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology
    • Octopamine

      2021, Handbook of Hormones: Comparative Endocrinology for Basic and Clinical Research
    View all citing articles on Scopus
    View full text